Wild mouse species and the various inbred laboratory mouse strains differ from one another in their susceptibility to the mouse gammaretroviruses and retrovirus-induced cancers. These differences are due to variations in specific host genes, and we have been engaged in an ongoing effort to identify and characterize several mouse genes involved in virus resistance. Our major interest has been on factors that interfere directly with virus infection and replication, and we focus our efforts on those factors that inhibit virus entry and the early post-entry stages of the virus replicative cycle. At the level of entry, there are two types of resistance genes that target the receptor-virus interaction. Receptors can be blocked by virus envelope glycoprotein produced by endogenous retroviruses, or resistance can be cause by polymorphisms in the cell surface receptor. After the gammaretrovirus enters the receptive cell, reverse transcription and translocation to the nucleus can be inhibited or altered by virus resistance factors Fv1, mApobec/Rfv3, and TRIM5alpha. Our current aim is to characterize these resistance factors and the viruses they target, define the origin and extent of antiviral activity in Mus evolution, and elucidate the responsible mechanisms. This work relies heavily on wild mice because laboratory strains provide only a limited sampling of the genetic diversity in Mus. Also, wild mouse species allow us to examine survival strategies in natural populations that harbor virus and to follow the evolution of the resistance genes. These mice additionally provide a source of novel resistance genes and virus variants. One set of projects is concerned with cell surface virus receptors and these studies focus on identifying viral and cell receptor determinants responsible for virus binding, entry, and receptor mediated cytopathicity. One series of experiments focuses on two unusual variants of the ecotropic gammaretroviruses that are cytopathic in M. dunni cells and also have altered host range. These phenotypes are due to different amino acid substitutions at the same site in viral envelope gene of the two viruses. This substitution alters one of the 3 amino acids that form the cell surface receptor binding site. The fact that these 2 viruses cause cytopathic effects in a cell line with a variant receptor gene suggests that the virus-receptor interaction mediates cytopathicity. This conclusion was confirmed by the observation that cytopathicity due to virus infection is seen in stable transfectants expressing this variant receptor but not in transfectants expressing the prototypical receptor. We have now inoculated mice with this cytopathic virus to determine if the lymphoma-type cancer induced by the progenitor virus is altered by the mutations that cause cytopathic cell fusion. Because evidence also suggests that cytopathicity is affected by inhibitors of glycosylation, we are currently evaluating the role of glycosylation in virus infectivity and cytopathicity. We have now demonstrated that receptor glycosylation modulates virus entry through both naturally occuring variants of the ecotropic receptor. We are currently looking at the effect of virus envelope glycosylation on the efficiency of virus entry. In another series of experiments, we have been characterizing virus susceptibility differences identified in a panel of cells derived from evolutionarily divergent wild mouse species. These cells show novel patterns of resistance not found among laboratory strains. One of these resistance phenotypes was identified in the African pygmy mouse, Mus minutoides which is completely resistant to the ecotropic gammaretrovirus found in common inbred strains, although it is susceptible to laboratory virus strains such as Moloney leukemia virus. We have shown that the block to virus replication is post entry and targets the viral capsid. We have now identified the responsible gene as Fv1, and we expanded this analysis to examine other wild mice for Fv1 variants. This phylogenetic analysis led us to identify 6 amino acid residues in Fv1 as being under positive evolutionary selection. 3 of these residues are known to be responsible for resistance in laboratory mice suggesting that this resistance has been important throughout mouse evolution. 3 additional selected residues are in the region of Fv1 related to the virus capsid, suggesting that Fv1 interferes with the structural integrity of infecting virions. Among the mouse genes responsible for resistance to mouse leukemia viruses is Rfv3 (recovery from Friend virus). This gene was recently shown to be encoded by the mouse APOBEC (mA3) gene, a cytidine deaminase gene known to restrict other retroviruses in mice and in humans. We sequenced mA3 from multiple laboratory and wild mice to examine its evolution. We discovered that the mA3 allele in virus resistant mice is disrupted by insertion of the regulatory signals of a mouse leukmia virus that may be responsible for enhanced mA3 expression and altered splicing. We also subjected the mA3 protein coding sequences to phylogenetic analysis. We identified 6 sites under positive selection, 6 of which are in two clusters that distinguish the virus restrictive and nonrestrictive mouse variants, and that are known to be important for human APOBEC3G function. We also showed that these two clusters are positioned opposite each other along the groove that forms the mA3 active site. We thus show that mA3 has had an antiviral role throughout mouse evolution, and we identify an inserted regulatory sequence and two clusters in the protein coding sequence that may contribute to this antiviral function. We are also interested in determining the extent to which virus resistance is mediated by polymorphisms of the cell surface receptor. We seek to analyze the XPR1 receptor for the xenotropic/polytropic gammaretroviruses. This family of viruses is widespread in mice, and related viruses have now been found in koalas, gibbon apes, and human cancer patients. We identified a fourth XPR1 susceptibility variant in the Asian mouse species, Mus pahari. These mice are resistant to the polytropic gammaretroviruses and susceptible to the xenotropic viruses, a pattern that is the opposite of that found in laboratory mice. We have now cloned and characterized the pahari cellular receptor that mediates entry of both xenotropic and polytropic viruses. We analyzed chimeras and mutants of the different receptors to show that 2 of the 4 extracellular loops of this membrane protein contain the determinants for virus entry. We identified two critical amino acids, one in each loop, that independently mediate entry of xenotropic viruses. We also identified 3 amino acids responsible for entry of the polytropic subgroup of viruses. Using cells expressing the 4 different XPR1 receptors and the 2 additional variants found in hamster and rat cells, we showed that there are in fact 6 host range subtypes of viruses that rely on this receptor for entry, and one of these variants is defined by the gammaretrovirus-like XMRV virus isolated from human prostate cancer patients. We are now characterizing this receptor in primates and in human cancer patients. The extent of this polymorphism in XPR1 and in the envelope genes should help us understand the entry process and may help account for the incidence of interspecies transmissions of these viruses in rodent and non-rodent species.